Influenza virus reassortment

ABSTRACT

Methods for producing reassortant viruses are provided wherein the transcription and/or translation of the hemagglutinin and/or neuraminidase genes are suppressed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase patent application ofPCT/IB2011/052218, filed May 20, 2011, which claims priority to U.S.Provisional patent application Ser. No. 61/396,110 filed, May 21, 2010,all of which are hereby incorporated by reference in the presentdisclosure in their entirety.

SUBMISSION OF SEQUENCE LISTING AS ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 223002126700SeqList.txt,date recorded: Nov. 20, 2012, size: 54 KB).

TECHNICAL FIELD

This invention is in the field of influenza virus reassortment.Furthermore, it relates to manufacturing vaccines for protecting againstinfluenza viruses.

BACKGROUND ART

The most efficient protection against influenza infection is vaccinationagainst the circulating strain and it is important to produce influenzaviruses for vaccine production as quickly as possible.

Wild-type influenza virus typically grows very slowly in eggs and cellculture. In order to obtain a faster-growing virus strain for vaccineproduction it is currently common practice to reassort the circulatinginfluenza strain (referred to herein as the vaccine strain) with afaster-growing high-yield backbone strain. This can be achieved byco-infecting cells in cell culture or the amniotic fluid of embryonatedhen eggs with the vaccine strain and the backbone strain. Antibodiesspecific for the backbone strain's hemagglutinin (HA) and neuraminidase(NA) proteins are then added to block influenza viruses which carry thebackbone strain's HA and/or NA protein from replicating. Over severalpassages of this treatment one can select for fast-growing reassortantinfluenza viruses which contain the HA and NA segments from the vaccinestrain and the other viral segments (i.e. those encoding PB1, PB2, PA,NP, M1, M2, NS1 and NS2) from the backbone strain.

The current approaches have several drawbacks. For example, it typicallytakes about 35 days from the arrival of a new influenza strain to obtainthe final high-growth reassortant, which causes delays in the productionof influenza vaccines. Furthermore, the need to passage the virusesseveral times increases the risk for mutations in the HA antigen tooccur which can result in an unwanted change of antigenicity. The use ofpolyclonal antisera to inhibit the propagation of non-reassorted virusesalso increases the risk of introducing adventitious viral agents andother contaminants.

It is an object of the invention to provide further and improved methodsfor producing reassortant influenza viruses.

SUMMARY OF PREFERRED EMBODIMENTS

The inventors have now surprisingly discovered that preferentiallyreducing the transcription and/or translation of the backbone strain'sHA and/or NA genes during virus production (e.g. by using RNAinhibition) can greatly increase the speed by which reassortant virusesare produced. The methods further have the advantage that they do notrely on the use of antibodies and so the use of animal derived productsmay be avoided. Furthermore, the likelihood of spontaneous mutations islower as fewer passages are necessary to obtain reassortant viruses.

The invention provides a method for preparing a reassortant influenzavirus comprising the steps of (i) infecting a culture host with a firstinfluenza strain and a second influenza strain; (ii) contacting theculture host of step (i) with an inhibitory agent wherein saidinhibitory agent preferentially reduces the transcription and/ortranslation of the hemagglutinin and/or neuraminidase genes of one ofthe influenza strains of (i); (iii) culturing the culture host in orderto produce reassortant virus and optionally (iv) purifying the virusobtained in step (iii).

The methods of the invention may further comprise steps of (v) infectinga culture host with the reassortant virus obtained in step (iii) or step(iv); (vi) culturing the host from step (v) to produce further virus;and optionally (vii) purifying virus obtained in step (vi).

The invention provides a method of preparing a reassortant influenzavirus comprising the steps of (i) infecting a culture host with a firstinfluenza virus strain having at least one target segment; (ii)introducing into the culture host one or more expression construct(s)encoding the target segment(s) from a second influenza virus strain;(iii) contacting the culture host with an inhibitory agent whichpreferentially reduces the transcription and/or translation of the firstinfluenza strain's target segment(s); (iv) culturing the culture host inorder to produce reassortant virus; and optionally (v) purifying thevirus obtained in step (iv).

The invention provides a cell comprising expression construct(s)encoding: (i) all eight viral segments of a first influenza A or B virusgenome; (ii) at least one target segment of a second influenza A or Bvirus genome, wherein the second influenza strain's target segment(s)differs in sequence from the target segment of the first influenzastrain; and (iii) an inhibitory agent wherein said inhibitory agentpreferentially reduces transcription and/or translation of the targetsegment(s) in the first influenza strain.

The invention also provides a method for preparing a reassortantinfluenza virus comprising a step of culturing this cell in order toproduce reassortant virus. The method may further comprise steps ofinfecting a culture host with the reassortant virus obtained from thecell, then culturing the host to produce further virus, and then(optionally) purifying the further virus obtained in this way.

The invention also provides a method for producing influenza virusescomprising the steps of (a) infecting a culture host with a reassortantvirus obtained by the methods of the invention; (b) culturing the hostfrom step (a) to produce the virus; and optionally (c) purifying thevirus obtained in step (b).

The invention also provides a method of preparing a vaccine, comprisingthe steps of (d) preparing a virus by the methods of any one of theembodiments described above and (e) preparing vaccine from the virus.

Influenza Strains

Influenza viruses are segmented negative strand RNA viruses. Influenza Aand B viruses have eight segments (PB2, PB1, PA, HA, NP, NA, M and NS),whereas influenza C virus has seven (no NA segment). The virus requiresthe presence of at least four viral proteins (PB1, PB2, PA andnucleoprotein) to initiate genome replication.

The methods of the invention use influenza backbone strain(s) andvaccine strain(s). The backbone strain and vaccine strain used willusually differ in one or more (e.g. 2, 3, 4, 5, 6, 7 or 8) viralsegments that can be differentially inhibited by inhibitory agents asdescribed below. The backbone strain is inhibited more than the vaccinestrain for the desired segment(s) and therefore production of areassortant strain is favoured.

The vaccine strains can be pandemic as well as inter-pandemic (seasonal)influenza strains. The vaccine strains may contain the influenza A virusHA subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14,H15 or H16. They may contain the influenza A virus NA subtypes N1, N2,N3, N4, N5, N6, N7, N8 or N9. Where the vaccine strain is a seasonalinfluenza strain, the vaccine strain may have a H1 or H3 subtype. In oneaspect of the invention the vaccine strain is a H1N1 or H3N2 strain.

The vaccine strains may also be pandemic strains or potentially pandemicstrains. The characteristics of an influenza strain that give it thepotential to cause a pandemic outbreak are: (a) it contains a newhemagglutinin compared to the hemagglutinins in currently-circulatinghuman strains, i.e. one that has not been evident in the humanpopulation for over a decade (e.g. H2), or has not previously been seenat all in the human population (e.g. H5, H6 or H9, that have generallybeen found only in bird populations), such that the human populationwill be immunologically naïve to the strain's hemagglutinin; (b) it iscapable of being transmitted horizontally in the human population; and(c) it is pathogenic to humans. A vaccine strain with H5 hemagglutinintype is preferred where the reassortant virus is used in vaccines forimmunizing against pandemic influenza, such as a H5N1 strain. Otherpossible strains include H5N3, H9N2, H2N2, H7N1 and H7N7, and any otheremerging potentially pandemic strains. The invention is particularlysuitable for producing reassortant viruses for use in vaccines forprotecting against potential pandemic virus strains that can or havespread from a non-human animal population to humans, for example aswine-origin H1N1 influenza strain.

The backbone strain can be any known influenza virus strain but it ispreferred that it is an influenza virus strain that grows quickly incells and/or the allantoic fluid of eggs. This is preferred becausereassortant influenza viruses are often produced in order to obtain afast growing influenza virus for vaccine production. Examples of suchbackbone strains include but are not limited to A/Puerto Rico/8/34,A/Ann Arbor/6/60 and B/Ann Arbor/1/66.

A reassortant influenza A virus produced according to the invention mayinclude one or more RNA segments from a A/PR/8/34 virus (typically 6segments from A/PR/8/34, with the HA and NA segments being from avaccine strain, i.e. a 6:2 reassortant). It may also include one or moreRNA segments from a A/WSN/33 virus, or from any other virus strainuseful for generating reassortant viruses for vaccine preparation. Areassortant influenza A virus may include fewer than 6 (i.e. 1, 2, 3, 4or 5) viral segments from an AA/6/60 influenza virus (A/Ann Arbor/6/60).A reassortant influenza B virus may include fewer than 6 (i.e. 1, 2, 3,4 or 5) viral segments from an AA/1/66 influenza virus (B/AnnArbor/1/66).

The reassortant influenza strain of the invention may comprise one ormore genome segments from an A/California/4/09 strain, preferably the HAsegment and/or the NA segment as these are the main vaccine antigens.Thus, for instance, the HA gene segment may encode a H1 hemagglutininwhich is more closely related to SEQ ID NO: 7 than to SEQ ID NO: 8 (i.e.has a higher degree sequence identity when compared to SEQ ID NO: 7 thanto SEQ ID NO: 8 using the same algorithm and parameters). SEQ ID NOs: 7and 8 are 80% identical. Similarly, the NA gene may encode a N1neuraminidase which is more closely related to SEQ ID NO: 9 than to SEQID NO: 10. SEQ ID NOs: 9 and 10 are 82% identical.

Reassortant influenza B virus can also be produced. Influenza B virusesdo not currently display different HA subtypes, but they do fall intotwo distinct lineages: B/Victoria/2/87-like and B/Yamagata/16/88-like.These lineages emerged in the late 1980s and have HAs which can beantigenically and/or genetically distinguished from each other [1]. Areassortant influenza B strain of the invention can comprise HA from aB/Victoria/2/87-like strain or a B/Yamagata/16/88-like strain.

Viral segments and sequences from the A/PR/8/34, A/AA/6/60, B/AA/1/66,and A/California/04/09 strains are widely available. Their sequences areavailable on the public databases e.g. GI:89779337, GI:89779334,GI:89779332, GI:89779320, GI:89779327, GI:89779325, GI:89779322,GI:89779329, see also SEQ ID NOs 1-6.

The choice of backbone strain can depend on the vaccine strain withwhich it is used. In general, it will be desirable to choose strainswhose HA and/or NA segments do not show a high degree of identity on thenucleic acid or amino acid level as this can make it easier to findinhibitory agents which preferentially reduce transcription and/ortranslation of the backbone strain's HA and/or NA segments. For examplethe degree of identity may be less than 99%, less than 95%, less than90%, less than 85%, less than 80% or less than 75%. The HA and/or NAviral segments of the backbone and the vaccine strain can be ofdifferent subtypes. For example, when a H3N2 strain is used as a vaccinestrain a backbone strain which has a H1N1 subtype (e.g. A/PR/8/34) canbe used or vice versa. It is also possible, however, to use a backbonestrain and a vaccine strain with the same HA and/or NA subtypes (e.g. aH1 vaccine strain and a H1 backbone strain) in the methods of theinvention provided that the transcription and/or translation of thebackbone strain's HA and/or NA genes can be preferentially reduced.

Inhibitory Agents

Suitable inhibitory agents for use in the invention are those which canpreferentially reduce the transcription and/or translation of thebackbone strain's HA and/or NA gene(s) relative to the vaccine strain'sHA and/or NA gene(s). The preferential reduction of the backbonestrain's HA and/or NA protein levels either at the transcriptional ortranslational level favours the formation of reassortant influenzaviruses because the likelihood increases that the HA and/or NA proteinsof the vaccine strain will be incorporated as their relative abundanceincreases.

Where the inhibitory agent is a transcriptional inhibitor, it will beconsidered to preferentially reduce transcription if it reducestranscription of the backbone strain's HA and/or NA gene(s) by at leastx+5% (e.g. x+10%, x+20%, x+30%, x+40%) provided that the inhibitoryagent reduces transcription of the vaccine strain's HA and/or NA genesby x % or less (e.g. x−5%, x−10%, x−20%, x−30% or x−40%), wherein thereduction is measured in comparison to a control sample which was nottreated with the inhibitory agent. In this context, x can be 0, 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or99. Thus, for example, where the transcription of the backbone strain'sHA gene is reduced by 30%, the transcription of the vaccine strain's HAshould be reduced by a maximum of 25%. Suitable methods for measuringthe transcriptional reduction of the genes by the inhibitory agent willbe evident to the skilled person. For example, two separate cellcultures can be infected with the influenza virus of interest. One ofthe infected cultures is contacted with the inhibitory agent of interestwhile the other infected culture is either not treated or treated with asubstance which is known not to reduce transcription of the HA and/or NAgene(s) of the influenza virus with which the culture was infected (forexample, phosphate buffer saline (PBS) or an inhibitory agent withspecificity for an unrelated gene). RNA can then be isolated from bothsamples, cDNA can be transcribed from the isolated RNA and real-time PCRcan be performed with the cDNA from both samples using primers specificfor the HA and/or NA gene(s) in order to compare the expression levelsof the genes in the presence and absence of the inhibitory agent.

A translational inhibitor will be considered to preferentially reducetranslation of the backbone strain's HA and/or NA gene(s) if it reducesthe backbone strain's HA and/or NA protein levels by at least y+5% (e.g.x+10%, x+20%, x+30%, x+40%) provided that the inhibitory agent reducestranslation of the vaccine strain's HA and/or NA genes by y % or less(e.g. x−5%, x−10%, x−20%, x−30% or x−40%), wherein the reduction ismeasured in comparison to a control sample which was not treated withthe inhibitory agent. In this context, y can be 0, 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99. Thus, forexample, where the translation of the backbone strain's HA gene isreduced by 30%, the transcription of the vaccine strain's HA should bereduced by a maximum of 25%. Suitable methods for measuring reduction oftranslation of the HA and/or NA genes will be evident to the skilledperson. For example, two separate cell cultures can be infected with theinfluenza virus. One of the infected cultures is contacted with theinhibitory agent of interest while the other infected culture is eithernot treated or treated with a substance which is known not to inhibittranslation of the HA and/or NA protein(s) of the influenza virus withwhich the culture was infected (for example, PBS or an inhibitory agentwith specificity for an unrelated gene). Proteins can be isolated fromboth samples and the protein levels of the HA and/or NA protein(s) canbe analysed and compared by quantitative western blot analysis (see, forexample, chapter 57 of reference 2).

Where more than one backbone strain is used in the methods of theinvention, an inhibitory agent will be suitable if it preferentiallyreduces the transcription and/or translation of the HA and/or NA genesof at least one of the backbone strains used.

It is not generally necessary to test the suitability of the inhibitoryagent(s) each time an influenza virus is reassorted in accordance withthe present invention as a backbone strain used for reassortment can beused for a variety of different vaccine strains. Thus, once a suitableinhibitory agent(s) for a particular backbone strain has beenidentified, it is possible to use the same agent(s) for all methodswhere that particular backbone strain is used, and it is necessary onlyto verify that the inhibitory agent preferentially reduces transcriptionand/or translation of the backbone strain's HA and/or NA genes relativeto the HA and/or NA genes of the vaccine strain which is used.

Suitable inhibitory agents will be known to the skilled person andinclude, but are not limited to, short interfering RNAs (siRNA),double-stranded RNAs (dsRNA), micro-RNAs (miRNAs), short hairpin RNAs(shRNA), or small interfering DNAs (siDNAs) like e.g., phosphorothioateoligomers (PSOs) or phosphorodiamidate morpholino oligomer (PMOs).

Short interfering RNAs (siRNAs) are particularly suitable for use in themethods of the present invention. This is because they can besynthesized quickly and cheaply and they can suppress expression of agene with high specificity. Even a difference of a single nucleotidebetween the target sequence and an off-target sequence can achievespecific silencing of the target sequence [3]. The use of siRNAs in eggshas, for example, been described in reference 4.

Methods for designing siRNAs that specifically silence a gene ofinterest are known to those skilled in the art. For example, variousprograms are available that permit the design of gene specific siRNAs[5, 6]. Examples of such siRNAs for use in the invention are shown inTable 1. The siRNA sequences HA1-HA24 and NA1-NA24 in Table 1 have beendesigned to differentially inhibit the HA and NA of A/PR/8/34 in thepresence of A/Perth/16/09.

The use of siDNAs in the methods of the invention is also preferred.These have the advantage that they are easier to synthesize, are morestable, are taken up more easily by the cell and act faster than siRNAswhile still showing a comparable specificity for the target sequence[7].

The use of siRNAs and siDNAs is specifically advantageous when thebackbone strain and the vaccine strain have a high degree of identity intheir viral segments that encode the HA and/or NA genes, e.g. when theyare from the same influenza virus subtype, as these inhibitors are knownto show high sequence specificity. In order to achieve preferentialreduction of the backbone strain's HA and/or NA protein levels comparedto the vaccine strain's HA and/or NA protein levels, the siRNAs and/orsiDNAs can be designed such that they target areas of sequencevariations between the HA and/or NA genes from the backbone strain andthe vaccine strain. Areas of sequence variation can be determined byaligning the sequences from the two strains.

Specific examples of siDNAs are PMOs. These are synthetic antisenseoligomers which are usually designed to bind to the translation startsite where they can interfere with progression of the ribosomalinitiation complex from the 5′ cap to the start codon. The advantage ofPMOs is that they are more stable compared to RNA or even DNA [8].Methods for designing PMOs are known in the art [9].

Other suitable inhibitory agents are PSOs. These are synthetic oligomerswherein an oxygen atom is replaced by a non-bridging sulfur in theoligophosphate backbone of the DNA. PSOs are advantageous as they aremore stable compared to unmodified DNA and RNA oligomers.

When shRNAs are used they are usually introduced into the culture hostas a DNA expression construct that can express the shRNA. These shRNAexpression constructs will typically contain a sequence encoding a siRNAmolecule and the reversed complementary sequence of the siRNA moleculeseparated by a short linker sequence on the same DNA strand. The siRNAsequence can be designed as described above and it is within the meansof the skilled person to identify the reverse complementary sequenceonce the siRNA sequence is known. Examples of DNA sequences that encodesuitable shRNAs for use in the invention are shown in Table 2. Thesequences in this table use ‘GGGGGGG’ as an exemplary linker sequencebut the skilled person can easily replace it with other suitablesequences.

It is also envisioned to use more than one kind of inhibitory agent inthe methods of the present invention.

The use of further inhibitory agents in addition to or instead of thosewith specificity for the HA and/or NA gene(s) of the backbone strain isalso within the scope of the present invention. For example, it ispossible to add one or more inhibitory agents which can preferentiallyreduce the transcription and/or translation of one or more of thevaccine strain's backbone segments. This has the advantage that thelikelihood of formation of desired reassortant viruses is furtherincreased. Suitable inhibitory agents can be identified by the samemeans mutatis mutandis as described above for inhibitory agents thatpreferentially reduce transcription and/or translation of the backbonestrain's HA and/or NA genes.

The inhibitory agent can be introduced into the individual cells in thecell culture or the egg (allantoic fluid) by any means known to those ofskill in the art. For example, they can be introduced byelectroporation, DEAE-dextran, calcium phosphate precipitation,liposomes, microinjection, or microparticle-bombardment.

Where the reassortant viruses are produced in cell culture, it ispossible to use cells which have been stably transfected with one ormore expression constructs encoding the inhibitory agent(s). This hasthe advantage that the same cell line can be used each time a particularbackbone strain is employed thus eliminating the need to separatelyintroduce inhibitory agent(s) each time the methods of the invention arepractised.

The one or more inhibitory agent(s) can be introduced into the host cellor the allantoic fluid before, during or after infection with theinfluenza virus(es).

Virus Reassortment

Reassortant influenza viruses are frequently produced by co-infecting aculture host, usually cell culture or eggs, with a backbone strain and avaccine strain. Reassortant viruses are selected by adding antibodieswith specificity for the HA and/or NA proteins of the backbone strain inorder to select for reassortant viruses that contain the vaccinestrain's HA and/or NA proteins. Over several passages of this treatmentone can select for fast-growing reassortant viruses containing thevaccine strain's HA and/or NA segments.

Reassortant influenza viruses between two, three or more differentinfluenza strains can be produced. The reassortant viruses producedcontain at least one (i.e. one, two, three, four, five or six) backboneviral segment from the backbone strain. The backbone viral segments arethose which do not encode HA or NA. Thus, backbone segments willtypically encode the PB1, PB2, PA, NP, M₁, M₂, NS₁ and NS₂ polypeptidesof the influenza virus. The reassortant viruses will not typicallycontain the segments encoding HA and NA from the backbone strain eventhough reassortant viruses which comprise either the HA or the NA butnot both from the backbone strain are also envisioned.

When the reassortant viruses are reassortants between two influenzastrains, the reassortant viruses will generally include segments fromthe backbone strain and the vaccine strain in a ratio of 1:7, 2:6, 3:5,4:4, 5:3, 6:2 or 7:1. Having a majority of segments from the backbonestrain, in particular a ratio of 6:2, is typical. When the reassortantviruses of the invention are reassortants of three strains, thereassortant virus will generally include segments from the backbonestrain, the vaccine strain and the third strain in a ratio of 1:1:6,1:2:5, 1:3:4, 1:4:3, 1:5:2, 1:6:1, 2:1:5, 2:2:4, 2:3:3, 2:4:2, 2:5:1,3:1:2, 3:2:1, 4:1:3, 4:2:2, 4:3:1, 5:1:2, 5:2:1 or 6:1:1. For example,the reassortant influenza strains may contain viral segments from morethan one backbone strain and/or more than one vaccine strain.

The ‘second influenza strain’ used in the methods of the invention isdifferent to the first influenza strain which means that one or more(e.g. 2, 3, 4, 5, 6, 7, or 8) of their viral segments will be different.

Reverse Genetics

The invention will usually be applied in the context of “traditional”reassortment techniques, but it can also be used in reverse genetics(RG) systems or in combinations of traditional reassortment techniquesand RG systems. A problem with some RG systems is that it can be hard tointroduce the required expression constructs into a culture host (due tolow transfection efficiency, for example) which can make the RG systeminefficient. The invention overcomes this problem by providing a methodwherein some of the viral segments for the reassortant influenza virusare provided by infecting the culture host with an influenza virus whileothers are provided on one or more expression constructs.

In the methods of the invention, a culture host is infected with a firstinfluenza A or B strain which has eight genome segments of which atleast one (for example, one, two, three, four, five, or six) are targetsegments. The target segment(s) from the first influenza strain arethose viral segments which will not be present in the reassortantinfluenza virus produced according to the methods of the invention. Thetarget segments of a second influenza virus strain are introduced on oneor more expression construct(s). The target segment(s) of the secondinfluenza strain are those segment(s) which will be present in thereassortant influenza virus produced according to the methods of theinvention. The transcription and/or translation of the first influenzastrain's target segment(s) is preferentially reduced by the inhibitoryagent. Typically, the first influenza strain will have one or two targetsegments (usually HA and/or NA) and accordingly one or two targetsegments from the second influenza strain are introduced on one or moreexpression construct(s). The inhibitory agent(s) may be encoded on thesame construct as the second influenza strain's segment(s) or ondifferent construct(s).

For example, when the vaccine strain's HA segment is introduced into theculture host on an expression construct and the culture host is infectedwith the backbone strain, the inhibitory agent will be specific for thebackbone strain's HA segment. Similarly, when the vaccine strain's NA isintroduced on the one or more expression construct(s), the inhibitoryagent will be specific for the backbone strain's NA segment. If both theHA and NA segments are introduced on the one or more expressionconstruct(s), the inhibitory agent will be specific for the backbonestrain's HA and NA segments. The target segment(s) will typically be HAand/or NA.

The viral segments introduced on the one or more expression construct(s)can be the HA and/or NA segments of the vaccine strain while thebackbone segments are provided by the influenza virus used to infect theculture host. In this embodiment, the culture host can be contacted withinhibitory agents which preferentially reduce transcription and/ortranslation of the backbone strain's HA and/or NA segments. It is alsopossible to provide one or more backbone segment(s) on the expressionconstruct(s).

The first and the second influenza virus are different. Furthermore, inthe methods of the invention, the steps of (i) infecting the culturehost with a virus, (ii) introducing the one or more expressionconstruct(s) into the culture host and (iii) contacting the culture hostwith an inhibitory agent can be performed in any order.

Furthermore, although most RG systems permit ready omission of abackbone strain's segments, in other systems this is not so easy. Forexample, some RG systems encode 6 viral segments on a first plasmid plusthe HA and NA genes on a second plasmid. It can thus be cumbersome tocreate a reassortant strain in which one of the first plasmid's segmentsis replaced, but the present invention overcomes this issue. Forexample, a RG system encoding a first strain can be modified to encode asegment from a second strain and also to inhibit the correspondingsegment for the first strain, thereby providing a reassortant in whichthis segment for the second strain replaces the first strain's.

Thus the invention provides a cell comprising expression construct(s)encoding: (i) all eight genome segments of a first influenza A or Bvirus genome; (ii) at least one target segment of a second influenza Aor B virus genome, wherein the second influenza strain's targetsegment(s) differ(s) in sequence from the target segment(s) of the firstinfluenza strain; and (iii) an inhibitory agent wherein said inhibitoryagent preferentially reduces transcription and/or translation of thetarget segment(s) in the first influenza strain.

The target segment will typically include HA and/or NA. For example, thecell may comprise expression construct(s) encoding: (i) all eightsegments of a backbone strain; (ii) at least a HA segment of a vaccinestrain influenza, wherein the vaccine strain's HA segment differs insequence from the backbone strain's HA segment; and (iii) an inhibitoryagent wherein said inhibitory agent preferentially reduces transcriptionand/or translation of the backbone strain's HA segment.

The cell can produce influenza virus containing a reassortant mixture ofbackbone and vaccine strain segments. Virus produced by the cell can beused for vaccine manufacture as described herein.

The backbone strain's segments will typically be encoded on a differentexpression construct from the vaccine strain's segment(s). Theinhibitory agent(s) may be encoded on the same construct as the vaccinestrain's segment(s) or on different construct(s). For example, a firstconstruct may encode all eight segments of an influenza virus. A secondconstruct can be added which encodes a vaccine strain's HA and NAsegments and which also encodes inhibitors of the backbone strain's HAand NA segments.

Viral RNA (vRNA) molecules can be expressed in a construct under thecontrol of, for example, pol I promoters, bacterial RNA polymerasepromoters, bacteriophage polymerase promoters, etc. Where certainproteins are required to form an infectious virus the RG system canprovide these proteins e.g. the system further comprises DNA moleculesthat encode viral proteins such that expression of both types of DNAleads to assembly of a complete infectious virus.

Culture Host

The culture host for use in the methods of the present invention can beembryonated hen eggs or cells.

The current standard method for influenza virus growth uses specificpathogen-free (SPF) embryonated hen eggs, with virus being purified fromthe egg contents (allantoic fluid). More recently, however, viruses havebeen grown in animal cell culture and, for reasons of speed and patientallergies, this growth method is preferred. If egg-based viral growth isused then one or more amino acids may be introduced into the allantoicfluid of the egg together with the virus [10].

When cells are used, the invention will typically use a cell linealthough, for example, primary cells may be used as an alternative. Thecell will typically be mammalian. Suitable mammalian cells of origininclude, but are not limited to, hamster, cattle, primate (includinghumans and monkeys) and dog cells. Various cell types may be used, suchas kidney cells, fibroblasts, retinal cells, lung cells, etc. Examplesof suitable hamster cells are the cell lines having the names BHK21 orHKCC. Suitable monkey cells are e.g. African green monkey cells, such askidney cells as in the Vero cell line [11-13]. Suitable dog cells aree.g. kidney cells, as in the CLDK and MDCK cell line. Thus suitable celllines include, but are not limited to: MDCK; CHO; 293T; BHK; Vero;MRC-5; PER.C6; WI-38; etc. Preferred mammalian cell lines for growinginfluenza viruses include: MDCK cells [14-17], derived from Madin Darbycanine kidney; Vero cells [18-20], derived from African green monkey(Cercopithecus aethiops) kidney; or PER.C6 cells [21], derived fromhuman embryonic retinoblasts. These cell lines are widely available e.g.from the American Type Cell Culture (ATCC) collection [22], from theCoriell Cell Repositories [23], or from the European Collection of CellCultures (ECACC). For example, the ATCC supplies various different Verocells under catalog numbers CCL-81, CCL-81.2, CRL-1586 and CRL-1587, andit supplies MDCK cells under catalog number CCL-34. PER.C6 is availablefrom the ECACC under deposit number 96022940. As a less-preferredalternative to mammalian cell lines, virus can be grown on avian celllines [e.g. refs. 24-26], including cell lines derived from ducks (e.g.duck retina) or hens e.g. chicken embryo fibroblasts (CEF), etc.Examples include avian embryonic stem cells [24,27], including the EBxcell line derived from chicken embryonic stem cells, EB45, EB14, andEB14-074 [28].

Preferred cells for use in the invention are MDCK cells [29-31], derivedfrom Madin Darby canine kidney. The original MDCK cells are availablefrom the ATCC as CCL-34. Derivatives of MDCK cells may also be used. Forinstance, reference 14 discloses a MDCK cell line that was adapted forgrowth in suspension culture (‘MDCK 33016’, deposited as DSM ACC 2219).Similarly, reference 32 discloses a MDCK-derived cell line that grows insuspension in serum-free culture (‘B-702’, deposited as FERM BP-7449).Reference 33 discloses non-tumorigenic MDCK cells, including ‘MDCK-S’(ATCC PTA-6500), ‘MDCK-SF101’ (ATCC PTA-6501), ‘MDCK-SF102’ (ATCCPTA-6502) and ‘MDCK-SF103’ (PTA-6503). Reference 34 discloses MDCK celllines with high susceptibility to infection, including ‘MDCK.5F1’ cells(ATCC CRL-12042). Any of these MDCK cell lines can be used.

For growth on a cell line, such as on MDCK cells, virus may be grown oncells in suspension [14,35,36] or in adherent culture. One suitable MDCKcell line for suspension culture is MDCK 33016 (deposited as DSM ACC2219). As an alternative, microcarrier culture can be used.

Cell lines supporting influenza virus replication are preferably grownin serum-free culture media and/or protein free media. A medium isreferred to as a serum-free medium in the context of the presentinvention if it contains no additives from serum of human or animalorigin. Protein-free is understood to mean cultures in whichmultiplication of the cells occurs with exclusion of proteins, growthfactors, other protein additives and non-serum proteins, but canoptionally include proteins such as trypsin or other proteases that maybe necessary for viral growth. The cells growing in such culturesnaturally contain proteins themselves.

Cell lines supporting influenza virus replication are preferably grownbelow 37° C. [37] (e.g. 30-36° C., or at about 30° C., 31° C., 32° C.,33° C., 34° C., 35° C., 36° C.), for example during viral replication.

Where virus is grown on a cell line then the growth culture, and alsothe viral inoculum used to start the culture, is preferably free from(i.e. will have been tested for and given a negative result forcontamination by) herpes simplex virus, respiratory syncytial virus,parainfluenza virus 3, SARS coronavirus, adenovirus, rhinovirus,reoviruses, polyomaviruses, birnaviruses, circoviruses, and/orparvoviruses [38].

Where virus has been grown on a mammalian cell line then the compositionwill advantageously be free from egg proteins (e.g. ovalbumin andovomucoid) and from chicken DNA, thereby reducing allergenicity. Theavoidance of allergens is useful for minimizing Th2 responses.

Virus Preparation

In a further aspect, the invention provides a method for the preparationof an influenza virus.

Where cells are used as a culture host in the methods of the invention,it is known that cell culture conditions (e.g. temperature, celldensity, pH value, etc.) are variable over a wide range subject to thecell line and the influenza virus strain employed and can be adapted tothe requirements of the application. The following information thereforemerely represents guidelines.

Cells are preferably cultured in serum-free or protein-free media.

Multiplication of the cells can be conducted in accordance with methodsknown to those of skill in the art. For example, the cells can becultivated in a perfusion system using ordinary support methods likecentrifugation or filtration. Moreover, the cells can be multipliedaccording to the invention in a fed-batch system before infection. Inthe context of the present invention, a culture system is referred to asa fed-batch system in which the cells are initially cultured in a batchsystem and depletion of nutrients (or part of the nutrients) in themedium is compensated by controlled feeding of concentrated nutrients.It can be advantageous to adjust the pH value of the medium duringmultiplication of cells before infection to a value between pH 6.6 andpH 7.8 and especially between a value between pH 7.2 and pH 7.3.Culturing of cells preferably occurs at a temperature between 30 and 40°C. After infection with the influenza viruses, the cells are preferablycultured at a temperature of between 30° C. and 36° C. or between 32° C.and 34° C. or at about 33° C. This is particularly preferred as it hasbeen shown that incubation of infected cells in this temperature rangeresults in production of a virus that results in improved efficacy whenformulated into a vaccine [39].

The oxygen partial pressure can be adjusted during culturing beforeinfection preferably at a value between 25% and 95% and especially at avalue between 35% and 60%. The values for the oxygen partial pressurestated in the context of the invention are based on saturation of air.Infection of cells occurs at a cell density of preferably about 8−25×10⁵cells/mL in the batch system or preferably about 5−20×10⁶ cells/mL inthe perfusion system. The cells can be infected with a viral dose (MOIvalue, “multiplicity of infection”; corresponds to the number of virusunits per cell at the time of infection) between 10⁻⁸ and 10, preferablybetween 0.0001 and 0.5.

Virus may be grown on cells in adherent culture or in suspension.Microcarrier cultures can be used. The cells may also be adapted forgrowth in suspension.

The methods according to the invention can include harvesting andisolation of viruses or the proteins generated by them. During isolationof viruses or proteins, the cells are separated from the culture mediumby standard methods like separation, filtration or ultrafiltration. Theviruses or the proteins are then concentrated according to methodssufficiently known to those skilled in the art, like gradientcentrifugation, filtration, precipitation, chromatography, etc., andthen purified. It is preferred that the viruses are inactivated duringor after purification. Virus inactivation can occur, for example, byβ-propiolactone or formaldehyde at any point within the purificationprocess.

Vaccine

The invention utilises virus produced according to the method to producevaccines.

Influenza vaccines are generally based either on live virus or oninactivated virus. Inactivated vaccines may be based on whole virions,‘split’ virions, or on purified surface antigens. Antigens can also bepresented in the form of virosomes. The invention can be used formanufacturing any of these types of vaccine.

Where an inactivated influenza virus is used, the vaccine may comprisewhole virion, split virion, or purified surface antigens (includinghemagglutinin and, usually, also including neuraminidase). Chemicalmeans for inactivating a virus include treatment with an effectiveamount of one or more of the following agents: detergents, formaldehyde,β-propiolactone, methylene blue, psoralen, carboxyfullerene (C60),binary ethylamine, acetyl ethyleneimine, or combinations thereof.Non-chemical methods of viral inactivation are known in the art, such asfor example UV light or gamma irradiation.

Virions can be harvested from virus-containing fluids, e.g. allantoicfluid or cell culture supernatant, by various methods. For example, apurification process may involve zonal centrifugation using a linearsucrose gradient solution (that optionally includes detergent to disruptthe virions) or affinity chromatography methods. Antigens may then bepurified, after optional dilution, by diafiltration.

Split virions are obtained by treating purified virions with detergents(e.g. ethyl ether, polysorbate 80, deoxycholate, tri-N-butyl phosphate,Triton X-100, Triton N101, cetyltrimethylammonium bromide, Tergitol NP9,etc.) to produce subvirion preparations, including the ‘Tween-ether’splitting process. Methods of splitting influenza viruses, for exampleare well known in the art e.g. see refs. 40-45, etc. Splitting of thevirus is typically carried out by disrupting or fragmenting whole virus,whether infectious or non-infectious with a disrupting concentration ofa splitting agent. The disruption results in a full or partialsolubilisation of the virus proteins, altering the integrity of thevirus. Preferred splitting agents are non-ionic and ionic (e.g.cationic) surfactants e.g. alkylglycosides, alkylthioglycosides, acylsugars, sulphobetaines, betains, polyoxyethylenealkylethers,N,N-dialkyl-Glucamides, Hecameg, alkylphenoxy-polyethoxyethanols, NP9,quaternary ammonium compounds, sarcosyl, CTABs (cetyl trimethyl ammoniumbromides), tri-N-butyl phosphate, Cetavlon, myristyltrimethylammoniumsalts, lipofectin, lipofectamine, and DOT-MA, the octyl- or nonylphenoxypolyoxyethanols (e.g. the Triton surfactants, such as Triton X-100 orTriton N101), polyoxyethylene sorbitan esters (the Tween surfactants),polyoxyethylene ethers, polyoxyethlene esters, etc. One useful splittingprocedure uses the consecutive effects of sodium deoxycholate andformaldehyde, and splitting can take place during initial virionpurification (e.g. in a sucrose density gradient solution). Thus asplitting process can involve clarification of the virion-containingmaterial (to remove non-virion material), concentration of the harvestedvirions (e.g. using an adsorption method, such as CaHPO₄ adsorption),separation of whole virions from non-virion material, splitting ofvirions using a splitting agent in a density gradient centrifugationstep (e.g. using a sucrose gradient that contains a splitting agent suchas sodium deoxycholate), and then filtration (e.g. ultrafiltration) toremove undesired materials. Split virions can usefully be resuspended insodium phosphate-buffered isotonic sodium chloride solution. Examples ofsplit influenza vaccines are the BEGRIVAC™, FLUARIX™, FLUZONE™ andFLUSHIELD™ products.

Purified influenza virus surface antigen vaccines comprise the surfaceantigens hemagglutinin and, typically, also neuraminidase. Processes forpreparing these proteins in purified form are well known in the art. TheFLUVIRIN™, AGRIPPAL™ and INFLUVAC™ products are influenza subunitvaccines.

Another form of inactivated antigen is the virosome [46] (nucleic acidfree viral-like liposomal particles). Virosomes can be prepared bysolubilization of virus with a detergent followed by removal of thenucleocapsid and reconstitution of the membrane containing the viralglycoproteins. An alternative method for preparing virosomes involvesadding viral membrane glycoproteins to excess amounts of phospholipids,to give liposomes with viral proteins in their membrane.

The method of the invention may also be used to produce live vaccines.Such vaccines are usually prepared by purifying virions fromvirion-containing fluids. For example, the fluids may be clarified bycentrifugation, and stabilized with buffer (e.g. containing sucrose,potassium phosphate, and monosodium glutamate). Various forms ofinfluenza virus vaccine are currently available (e.g. see chapters 17 &18 of reference 47). Live virus vaccines include Medlmmune's FLUMIST™product (trivalent live virus vaccine).

The virus may be attenuated. The virus may be temperature-sensitive. Thevirus may be cold-adapted. These three features are particularly usefulwhen using live virus as an antigen.

HA is the main immunogen in current inactivated influenza vaccines, andvaccine doses are standardised by reference to HA levels, typicallymeasured by SRID. Existing vaccines typically contain about 15 μg of HAper strain, although lower doses can be used e.g. for children, or inpandemic situations, or when using an adjuvant. Fractional doses such as½ (i.e. 7.5 μg HA per strain), ¼ and ⅛ have been used, as have higherdoses (e.g. 3× or 9× doses [48,49]). Thus vaccines may include between0.1 and 150 μg of HA per influenza strain, preferably between 0.1 and 50μg e.g. 0.1-20 μg, 0.1-15 μg, 0.1-10 μg, 0.5-5 μg, etc. Particular dosesinclude e.g. about 45, about 30, about 15, about 10, about 7.5, about 5,about 3.8, about 3.75, about 1.9, about 1.5, etc. per strain.

For live vaccines, dosing is measured by median tissue cultureinfectious dose (TCID₅₀) rather than HA content, and a TCID₅₀ of between10⁶ and 10⁸ (preferably between 10^(6.5)-10^(7.5)) per strain istypical.

Influenza strains used with the invention may have a natural HA as foundin a wild-type virus, or a modified HA. For instance, it is known tomodify HA to remove determinants (e.g. hyper-basic regions around theHA1/HA2 cleavage site) that cause a virus to be highly pathogenic inavian species. The use of reverse genetics facilitates suchmodifications.

Influenza strains used with the invention may have a natural HA as foundin a wild-type virus, or a modified HA. For instance, it is known tomodify HA to remove determinants (e.g. hyper-basic regions around theHA1/HA2 cleavage site) that cause a virus to be highly pathogenic inavian species. The use of reverse genetics facilitates suchmodifications.

As well as being suitable for immunizing against inter-pandemic strains,the compositions of the invention are particularly useful for immunizingagainst pandemic or potentially-pandemic strains. The invention issuitable for vaccinating humans as well as non-human animals.

Other strains whose antigens can usefully be included in thecompositions are strains which are resistant to antiviral therapy (e.g.resistant to oseltamivir [50] and/or zanamivir), including resistantpandemic strains [51].

Compositions of the invention may include antigen(s) from one or more(e.g. 1, 2, 3, 4 or more) influenza virus strains, including influenza Avirus and/or influenza B virus. Where a vaccine includes more than onestrain of influenza, the different strains are typically grownseparately and are mixed after the viruses have been harvested andantigens have been prepared. Thus a process of the invention may includethe step of mixing antigens from more than one influenza strain. Atrivalent vaccine is typical, including antigens from two influenza Avirus strains and one influenza B virus strain. A tetravalent vaccine isalso useful [52], including antigens from two influenza A virus strainsand two influenza B virus strains, or three influenza A virus strainsand one influenza B virus strain.

Pharmaceutical Compositions

Vaccine compositions manufactured according to the invention arepharmaceutically acceptable. They usually include components in additionto the antigens e.g. they typically include one or more pharmaceuticalcarrier(s) and/or excipient(s) (a thorough discussion of such componentsis available in reference 53). As described below, adjuvants may also beincluded.

Vaccine compositions will generally be in aqueous form. However, somevaccines may be in dry form, e.g. in the form of injectable solids ordried or polymerized preparations on a patch.

Vaccine compositions may include preservatives such as thiomersal or2-phenoxyethanol. It is preferred, however, that the vaccine should besubstantially free from (i.e. less than 5 μg/ml) mercurial material e.g.thiomersal-free [44,54]. Vaccines containing no mercury are morepreferred. An α-tocopherol succinate can be included as an alternativeto mercurial compounds [44]. Preservative-free vaccines are particularlypreferred.

To control tonicity, it is preferred to include a physiological salt,such as a sodium salt. Sodium chloride (NaCl) is preferred, which may bepresent at between 1 and 20 mg/ml. Other salts that may be presentinclude potassium chloride, potassium dihydrogen phosphate, disodiumphosphate dehydrate, magnesium chloride, calcium chloride, etc.

Vaccine compositions will generally have an osmolality of between 200mOsm/kg and 400 mOsm/kg, preferably between 240-360 mOsm/kg, and willmore preferably fall within the range of 290-310 mOsm/kg. Osmolality haspreviously been reported not to have an impact on pain caused byvaccination [55], but keeping osmolality in this range is neverthelesspreferred.

Vaccine compositions may include one or more buffers. Typical buffersinclude: a phosphate buffer; a Tris buffer; a borate buffer; a succinatebuffer; a histidine buffer (particularly with an aluminum hydroxideadjuvant); or a citrate buffer. Buffers will typically be included inthe 5-20 mM range.

The pH of a vaccine composition will generally be between 5.0 and 8.1,and more typically between 6.0 and 8.0 e.g. 6.5 and 7.5, or between 7.0and 7.8. A process of the invention may therefore include a step ofadjusting the pH of the bulk vaccine prior to packaging.

The vaccine composition is preferably sterile. The vaccine compositionis preferably non-pyrogenic e.g. containing <1 EU (endotoxin unit, astandard measure) per dose, and preferably <0.1 EU per dose. The vaccinecomposition is preferably gluten-free.

Vaccine compositions of the invention may include detergent e.g. apolyoxyethylene sorbitan ester surfactant (known as ‘Tweens’), anoctoxynol (such as octoxynol-9 (Triton X-100) ort-octylphenoxypolyethoxyethanol), a cetyl trimethyl ammonium bromide(‘CTAB’), or sodium deoxycholate, particularly for a split or surfaceantigen vaccine. The detergent may be present only at trace amounts.Thus the vaccine may include less than 1 mg/ml of each of octoxynol-10and polysorbate 80. Other residual components in trace amounts could beantibiotics (e.g. neomycin, kanamycin, polymyxin B).

A vaccine composition may include material for a single immunisation, ormay include material for multiple immunisations (i.e. a ‘multidose’kit). The inclusion of a preservative is preferred in multidosearrangements. As an alternative (or in addition) to including apreservative in multidose compositions, the compositions may becontained in a container having an aseptic adaptor for removal ofmaterial.

Influenza vaccines are typically administered in a dosage volume ofabout 0.5 ml, although a half dose (i.e. about 0.25 ml) may beadministered to children.

Compositions and kits are preferably stored at between 2° C. and 8° C.They should not be frozen. They should ideally be kept out of directlight.

Host Cell DNA

Where virus has been isolated and/or grown on a cell line, it isstandard practice to minimize the amount of residual cell line DNA inthe final vaccine, in order to minimize any potential oncogenic activityof the DNA.

Thus a vaccine composition prepared according to the inventionpreferably contains less than 10 ng (preferably less than 1 ng, and morepreferably less than 100 pg) of residual host cell DNA per dose,although trace amounts of host cell DNA may be present.

It is preferred that the average length of any residual host cell DNA isless than 500 bp e.g. less than 400 bp, less than 300 bp, less than 200bp, less than 100 bp, etc.

Contaminating DNA can be removed during vaccine preparation usingstandard purification procedures e.g. chromatography, etc. Removal ofresidual host cell DNA can be enhanced by nuclease treatment e.g. byusing a DNase. A convenient method for reducing host cell DNAcontamination is disclosed in references 56 & 57, involving a two-steptreatment, first using a DNase (e.g. Benzonase), which may be usedduring viral growth, and then a cationic detergent (e.g. CTAB), whichmay be used during virion disruption. Treatment with an alkylatingagent, such as β-propiolactone, can also be used to remove host cellDNA, and advantageously may also be used to inactivate virions [58].

Adjuvants

Compositions of the invention may advantageously include an adjuvant,which can function to enhance the immune responses (humoral and/orcellular) elicited in a subject who receives the composition. Preferredadjuvants comprise oil-in-water emulsions. Various such adjuvants areknown, and they typically include at least one oil and at least onesurfactant, with the oil(s) and surfactant(s) being biodegradable(metabolisable) and biocompatible. The oil droplets in the emulsion aregenerally less than 5 μm in diameter, and ideally have a sub-microndiameter, with these small sizes being achieved with a microfluidiser toprovide stable emulsions. Droplets with a size less than 220 nm arepreferred as they can be subjected to filter sterilization.

The emulsion can comprise oils such as those from an animal (such asfish) or vegetable source. Sources for vegetable oils include nuts,seeds and grains. Peanut oil, soybean oil, coconut oil, and olive oil,the most commonly available, exemplify the nut oils. Jojoba oil can beused e.g. obtained from the jojoba bean. Seed oils include saffloweroil, cottonseed oil, sunflower seed oil, sesame seed oil and the like.In the grain group, corn oil is the most readily available, but the oilof other cereal grains such as wheat, oats, rye, rice, teff, triticaleand the like may also be used. 6-10 carbon fatty acid esters of glyceroland 1,2-propanediol, while not occurring naturally in seed oils, may beprepared by hydrolysis, separation and esterification of the appropriatematerials starting from the nut and seed oils. Fats and oils frommammalian milk are metabolizable and may therefore be used in thepractice of this invention. The procedures for separation, purification,saponification and other means necessary for obtaining pure oils fromanimal sources are well known in the art. Most fish containmetabolizable oils which may be readily recovered. For example, codliver oil, shark liver oils, and whale oil such as spermaceti exemplifyseveral of the fish oils which may be used herein. A number of branchedchain oils are synthesized biochemically in 5-carbon isoprene units andare generally referred to as terpenoids. Shark liver oil contains abranched, unsaturated terpenoids known as squalene,2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene, which isparticularly preferred herein. Squalane, the saturated analog tosqualene, is also a preferred oil. Fish oils, including squalene andsqualane, are readily available from commercial sources or may beobtained by methods known in the art. Another preferred oil isα-tocopherol (see below).

Mixtures of oils can be used.

Surfactants can be classified by their ‘HLB’ (hydrophile/lipophilebalance). Preferred surfactants of the invention have a HLB of at least10, preferably at least 15, and more preferably at least 16. Theinvention can be used with surfactants including, but not limited to:the polyoxyethylene sorbitan esters surfactants (commonly referred to asthe Tweens), especially polysorbate 20 and polysorbate 80; copolymers ofethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO),sold under the DOWFAX™ tradename, such as linear EO/PO block copolymers;octoxynols, which can vary in the number of repeating ethoxy(oxy-1,2-ethanediyl) groups, with octoxynol-9 (Triton X-100, ort-octylphenoxypolyethoxyethanol) being of particular interest;(octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipidssuch as phosphatidylcholine (lecithin); nonylphenol ethoxylates, such asthe Tergitol™ NP series; polyoxyethylene fatty ethers derived fromlauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants),such as triethyleneglycol monolauryl ether (Brij 30); and sorbitanesters (commonly known as the SPANs), such as sorbitan trioleate (Span85) and sorbitan monolaurate. Non-ionic surfactants are preferred.Preferred surfactants for including in the emulsion are Tween 80(polyoxyethylene sorbitan monooleate), Span 85 (sorbitan trioleate),lecithin and Triton X-100.

Mixtures of surfactants can be used e.g. Tween 80/Span 85 mixtures. Acombination of a polyoxyethylene sorbitan ester such as polyoxyethylenesorbitan monooleate (Tween 80) and an octoxynol such ast-octylphenoxypolyethoxyethanol (Triton X-100) is also suitable. Anotheruseful combination comprises laureth 9 plus a polyoxyethylene sorbitanester and/or an octoxynol.

Preferred amounts of surfactants (% by weight) are: polyoxyethylenesorbitan esters (such as Tween 80) 0.01 to 1%, in particular about 0.1%;octyl- or nonylphenoxy polyoxyethanols (such as Triton X-100, or otherdetergents in the Triton series) 0.001 to 0.1%, in particular 0.005 to0.02%; polyoxyethylene ethers (such as laureth 9) 0.1 to 20%, preferably0.1 to 10% and in particular 0.1 to 1% or about 0.5%.

Where the vaccine contains a split virus, it is preferred that itcontains free surfactant in the aqueous phase. This is advantageous asthe free surfactant can exert a ‘splitting effect’ on the antigen,thereby disrupting any unsplit virions and/or virion aggregates thatmight otherwise be present. This can improve the safety of split virusvaccines [59].

Preferred emulsions have an average droplets size of <1 μm e.g. ≦750 nm,≦500 nm, ≦400 nm, ≦300 nm, ≦250 nm, ≦220 nm, ≦200 nm, or smaller. Thesedroplet sizes can conveniently be achieved by techniques such asmicrofluidisation.

Specific oil-in-water emulsion adjuvants useful with the inventioninclude, but are not limited to:

-   -   A submicron emulsion of squalene, Tween 80, and Span 85. The        composition of the emulsion by volume can be about 5% squalene,        about 0.5% polysorbate 80 and about 0.5% Span 85. In weight        terms, these ratios become 4.3% squalene, 0.5% polysorbate 80        and 0.48% Span 85. This adjuvant is known as ‘MF59’ [60-62], as        described in more detail in Chapter 10 of ref. 63 and chapter 12        of ref. 64. The MF59 emulsion advantageously includes citrate        ions e.g. 10 mM sodium citrate buffer.    -   An emulsion of squalene, DL-α-tocopherol, and polysorbate 80        (Tween 80). The emulsion may include phosphate buffered saline.        It may also include Span 85 (e.g. at 1%) and/or lecithin. These        emulsions may have from 2 to 10% squalene, from 2 to 10%        tocopherol and from 0.3 to 3% Tween 80, and the weight ratio of        squalene:tocopherol is preferably ≦1 as this provides a more        stable emulsion. Squalene and Tween 80 may be present volume        ratio of about 5:2 or at a weight ratio of about 11:5. One such        emulsion can be made by dissolving Tween 80 in PBS to give a 2%        solution, then mixing 90 ml of this solution with a mixture of        (5 g of DL-α-tocopherol and 5 ml squalene), then microfluidising        the mixture. The resulting emulsion may have submicron oil        droplets e.g. with an average diameter of between 100 and 250        nm, preferably about 180 nm. The emulsion may also include a        3-de-O-acylated monophosphoryl lipid A (3d-MPL). Another useful        emulsion of this type may comprise, per human dose, 0.5-10 mg        squalene, 0.5-11 mg tocopherol, and 0.1-4 mg polysorbate 80        [65].    -   An emulsion of squalene, a tocopherol, and a Triton detergent        (e.g. Triton X-100). The emulsion may also include a 3d-MPL (see        below). The emulsion may contain a phosphate buffer.    -   An emulsion comprising a polysorbate (e.g. polysorbate 80), a        Triton detergent (e.g. Triton X-100) and a tocopherol (e.g. an        α-tocopherol succinate). The emulsion may include these three        components at a mass ratio of about 75:11:10 (e.g. 750 μg/ml        polysorbate 80, 110 μg/ml Triton X-100 and 100 μg/ml        α-tocopherol succinate), and these concentrations should include        any contribution of these components from antigens. The emulsion        may also include squalene. The emulsion may also include a        3d-MPL (see below). The aqueous phase may contain a phosphate        buffer.    -   An emulsion of squalane, polysorbate 80 and poloxamer 401        (“Pluronic™ L121”). The emulsion can be formulated in phosphate        buffered saline, pH 7.4. This emulsion is a useful delivery        vehicle for muramyl dipeptides, and has been used with        threonyl-MDP in the “SAF-1” adjuvant [66] (0.05-1% Thr-MDP, 5%        squalane, 2.5% Pluronic L121 and 0.2% polysorbate 80). It can        also be used without the Thr-MDP, as in the “AF” adjuvant [67]        (5% squalane, 1.25% Pluronic L121 and 0.2% polysorbate 80).        Microfluidisation is preferred.    -   An emulsion comprising squalene, an aqueous solvent, a        polyoxyethylene alkyl ether hydrophilic nonionic surfactant        (e.g. polyoxyethylene (12) cetostearyl ether) and a hydrophobic        nonionic surfactant (e.g. a sorbitan ester or mannide ester,        such as sorbitan monoleate or ‘Span 80’). The emulsion is        preferably thermoreversible and/or has at least 90% of the oil        droplets (by volume) with a size less than 200 nm [68]. The        emulsion may also include one or more of: alditol; a        cryoprotective agent (e.g. a sugar, such as dodecylmaltoside        and/or sucrose); and/or an alkylpolyglycoside. The emulsion may        include a TLR4 agonist [69]. Such emulsions may be lyophilized.    -   An emulsion of squalene, poloxamer 105 and Abil-Care [70]. The        final concentration (weight) of these components in adjuvanted        vaccines are 5% squalene, 4% poloxamer 105 (pluronic polyol) and        2% Abil-Care 85 (Bis-PEG/PPG-16/16 PEG/PPG-16/16 dimethicone;        caprylic/capric triglyceride).    -   An emulsion having from 0.5-50% of an oil, 0.1-10% of a        phospholipid, and 0.05-5% of a non-ionic surfactant. As        described in reference 71, preferred phospholipid components are        phosphatidylcholine, phosphatidylethanolamine,        phosphatidylserine, phosphatidylinositol, phosphatidylglycerol,        phosphatidic acid, sphingomyelin and cardiolipin. Submicron        droplet sizes are advantageous.    -   A submicron oil-in-water emulsion of a non-metabolisable oil        (such as light mineral oil) and at least one surfactant (such as        lecithin, Tween 80 or Span 80). Additives may be included, such        as QuilA saponin, cholesterol, a saponin-lipophile conjugate        (such as GPI-0100, described in reference 72, produced by        addition of aliphatic amine to desacylsaponin via the carboxyl        group of glucuronic acid), dimethyldioctadecylammonium bromide        and/or N,N-dioctadecyl-N,N-bis(2-hydroxyethyl)propanediamine.    -   An emulsion in which a saponin (e.g. QuilA or QS21) and a sterol        (e.g. a cholesterol) are associated as helical micelles [73].    -   An emulsion comprising a mineral oil, a non-ionic lipophilic        ethoxylated fatty alcohol, and a non-ionic hydrophilic        surfactant (e.g. an ethoxylated fatty alcohol and/or        polyoxyethylene-polyoxypropylene block copolymer) [74].    -   An emulsion comprising a mineral oil, a non-ionic hydrophilic        ethoxylated fatty alcohol, and a non-ionic lipophilic surfactant        (e.g. an ethoxylated fatty alcohol and/or        polyoxyethylene-polyoxypropylene block copolymer) [74].

In some embodiments an emulsion may be mixed with antigenextemporaneously, at the time of delivery, and thus the adjuvant andantigen may be kept separately in a packaged or distributed vaccine,ready for final formulation at the time of use. In other embodiments anemulsion is mixed with antigen during manufacture, and thus thecomposition is packaged in a liquid adjuvanted form. The antigen willgenerally be in an aqueous form, such that the vaccine is finallyprepared by mixing two liquids. The volume ratio of the two liquids formixing can vary (e.g. between 5:1 and 1:5) but is generally about 1:1.Where concentrations of components are given in the above descriptionsof specific emulsions, these concentrations are typically for anundiluted composition, and the concentration after mixing with anantigen solution will thus decrease.

Packaging of Vaccine Compositions

Suitable containers for compositions of the invention (or kitcomponents) include vials, syringes (e.g. disposable syringes), nasalsprays, etc. These containers should be sterile.

Where a composition/component is located in a vial, the vial ispreferably made of a glass or plastic material. The vial is preferablysterilized before the composition is added to it. To avoid problems withlatex-sensitive patients, vials are preferably sealed with a latex-freestopper, and the absence of latex in all packaging material ispreferred. The vial may include a single dose of vaccine, or it mayinclude more than one dose (a ‘multidose’ vial) e.g. 10 doses. Preferredvials are made of colourless glass.

A vial can have a cap (e.g. a Luer lock) adapted such that a pre-filledsyringe can be inserted into the cap, the contents of the syringe can beexpelled into the vial (e.g. to reconstitute lyophilised materialtherein), and the contents of the vial can be removed back into thesyringe. After removal of the syringe from the vial, a needle can thenbe attached and the composition can be administered to a patient. Thecap is preferably located inside a seal or cover, such that the seal orcover has to be removed before the cap can be accessed. A vial may havea cap that permits aseptic removal of its contents, particularly formultidose vials.

Where a component is packaged into a syringe, the syringe may have aneedle attached to it. If a needle is not attached, a separate needlemay be supplied with the syringe for assembly and use. Such a needle maybe sheathed. Safety needles are preferred. 1-inch 23-gauge, 1-inch25-gauge and ⅝-inch 25-gauge needles are typical. Syringes may beprovided with peel-off labels on which the lot number, influenza seasonand expiration date of the contents may be printed, to facilitate recordkeeping. The plunger in the syringe preferably has a stopper to preventthe plunger from being accidentally removed during aspiration. Thesyringes may have a latex rubber cap and/or plunger. Disposable syringescontain a single dose of vaccine. The syringe will generally have a tipcap to seal the tip prior to attachment of a needle, and the tip cap ispreferably made of a butyl rubber. If the syringe and needle arepackaged separately then the needle is preferably fitted with a butylrubber shield. Preferred syringes are those marketed under the tradename “Tip-Lok”™.

Containers may be marked to show a half-dose volume e.g. to facilitatedelivery to children. For instance, a syringe containing a 0.5 ml dosemay have a mark showing a 0.25 ml volume.

Where a glass container (e.g. a syringe or a vial) is used, then it ispreferred to use a container made from a borosilicate glass rather thanfrom a soda lime glass.

A kit or composition may be packaged (e.g. in the same box) with aleaflet including details of the vaccine e.g. instructions foradministration, details of the antigens within the vaccine, etc. Theinstructions may also contain warnings e.g. to keep a solution ofadrenaline readily available in case of anaphylactic reaction followingvaccination, etc.

Methods of Treatment, and Administration of the Vaccine

The invention provides a vaccine manufactured according to theinvention. These vaccine compositions are suitable for administration tohuman or non-human animal subjects, such as pigs, and the inventionprovides a method of raising an immune response in a subject, comprisingthe step of administering a composition of the invention to the subject.The invention also provides a composition of the invention for use as amedicament, and provides the use of a composition of the invention forthe manufacture of a medicament for raising an immune response in asubject.

The immune response raised by these methods and uses will generallyinclude an antibody response, preferably a protective antibody response.Methods for assessing antibody responses, neutralising capability andprotection after influenza virus vaccination are well known in the art.Human studies have shown that antibody titers against hemagglutinin ofhuman influenza virus are correlated with protection (a serum samplehemagglutination-inhibition titer of about 30-40 gives around 50%protection from infection by a homologous virus) [75]. Antibodyresponses are typically measured by hemagglutination inhibition, bymicroneutralisation, by single radial immunodiffusion (SRID), and/or bysingle radial hemolysis (SRH). These assay techniques are well known inthe art.

Compositions of the invention can be administered in various ways. Themost preferred immunisation route is by intramuscular injection (e.g.into the arm or leg), but other available routes include subcutaneousinjection, intranasal [76-78], oral [79], intradermal [80,81],transcutaneous, transdermal [82], etc.

Vaccines prepared according to the invention may be used to treat bothchildren and adults. Influenza vaccines are currently recommended foruse in pediatric and adult immunisation, from the age of 6 months. Thusa human subject may be less than 1 year old, 1-5 years old, 5-15 yearsold, 15-55 years old, or at least 55 years old. Preferred subjects forreceiving the vaccines are the elderly (e.g. ≧50 years old, ≧60 yearsold, and preferably ≧65 years), the young (e.g. ≦5 years old),hospitalised subjects, healthcare workers, armed service and militarypersonnel, pregnant women, the chronically ill, immunodeficientsubjects, subjects who have taken an antiviral compound (e.g. anoseltamivir or zanamivir compound; see below) in the 7 days prior toreceiving the vaccine, people with egg allergies and people travellingabroad. The vaccines are not suitable solely for these groups, however,and may be used more generally in a population. For pandemic strains,administration to all age groups is preferred.

Preferred compositions of the invention satisfy 1, 2 or 3 of the CPMPcriteria for efficacy. In adults (18-60 years), these criteria are:(1)≧70% seroprotection; (2)≧40% seroconversion; and/or (3) a GMTincrease of ≧2.5-fold. In elderly (>60 years), these criteria are:(1)≧60% seroprotection; (2)≧30% seroconversion; and/or (3) a GMTincrease of ≧2-fold. These criteria are based on open label studies withat least 50 patients.

Treatment can be by a single dose schedule or a multiple dose schedule.Multiple doses may be used in a primary immunisation schedule and/or ina booster immunisation schedule. In a multiple dose schedule the variousdoses may be given by the same or different routes e.g. a parenteralprime and mucosal boost, a mucosal prime and parenteral boost, etc.Administration of more than one dose (typically two doses) isparticularly useful in immunologically naïve patients e.g. for peoplewho have never received an influenza vaccine before, or for vaccinatingagainst a new HA subtype (as in a pandemic outbreak). Multiple doseswill typically be administered at least 1 week apart (e.g. about 2weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about10 weeks, about 12 weeks, about 16 weeks, etc.).

Vaccines produced by the invention may be administered to patients atsubstantially the same time as (e.g. during the same medicalconsultation or visit to a healthcare professional or vaccinationcentre) other vaccines e.g. at substantially the same time as a measlesvaccine, a mumps vaccine, a rubella vaccine, a MMR vaccine, a varicellavaccine, a MMRV vaccine, a diphtheria vaccine, a tetanus vaccine, apertussis vaccine, a DTP vaccine, a conjugated H. influenzae type bvaccine, an inactivated poliovirus vaccine, a hepatitis B virus vaccine,a meningococcal conjugate vaccine (such as a tetravalent A-C-W135-Yvaccine), a respiratory syncytial virus vaccine, a pneumococcalconjugate vaccine, etc. Administration at substantially the same time asa pneumococcal vaccine and/or a meningococcal vaccine is particularlyuseful in elderly patients.

Similarly, vaccines of the invention may be administered to patients atsubstantially the same time as (e.g. during the same medicalconsultation or visit to a healthcare professional) an antiviralcompound, and in particular an antiviral compound active againstinfluenza virus (e.g. oseltamivir and/or zanamivir). These antiviralsinclude neuraminidase inhibitors, such as a(3R,4R,5S)-4-acetylamino-5-amino-3(1-ethylpropoxy)-1-cyclohexene-1-carboxylicacid or5-(acetylamino)-4-[(aminoiminomethyl)-amino]-2,6-anhydro-3,4,5-trideoxy-D-glycero-D-galactonon-2-enonicacid, including esters thereof (e.g. the ethyl esters) and salts thereof(e.g. the phosphate salts). A preferred antiviral is(3R,4R,5S)-4-acetylamino-5-amino-3(1-ethylpropoxy)-1-cyclohexene-1-carboxylicacid, ethyl ester, phosphate (1:1), also known as oseltamivir phosphate(TAMIFLU™).

General

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

The term “about” in relation to a numerical value x is optional andmeans, for example, x±10%.

Unless specifically stated, a process comprising a step of mixing two ormore components does not require any specific order of mixing. Thuscomponents can be mixed in any order. Where there are three componentsthen two components can be combined with each other, and then thecombination may be combined with the third component, etc.

Where animal (and particularly bovine) materials are used in the cultureof cells, they should be obtained from sources that are free fromtransmissible spongiform encephalopathies (TSEs), and in particular freefrom bovine spongiform encephalopathy (BSE). Overall, it is preferred toculture cells in the total absence of animal-derived materials.

Where a compound is administered to the body as part of a compositionthen that compound may alternatively be replaced by a suitable prodrug.

References to a percentage sequence identity between two amino acidsequences means that, when aligned, that percentage of amino acids arethe same in comparing the two sequences. This alignment and the percenthomology or sequence identity can be determined using software programsknown in the art, for example those described in section 7.7.18 ofreference 83. A preferred alignment is determined by the Smith-Watermanhomology search algorithm using an affine gap search with a gap openpenalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62. TheSmith-Waterman homology search algorithm is taught in reference 84.

References to a percentage sequence identity between two nucleic acidsequences mean that, when aligned, that percentage of bases are the samein comparing the two sequences. This alignment and the percent homologyor sequence identity can be determined using software programs known inthe art, for example those described in section 7.7.18 of reference 83.A preferred alignment program is GCG Gap (Genetics Computer Group,Wisconsin, Suite Version 10.1), preferably using default parameters,which are as follows: open gap=3; extend gap=1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 show the viral titer after incubating with the indicatedsiRNAs. FIG. 1 shows the viral titer of A/PR8/34 and FIG. 2 shows theviral titer of A/Victoria (H3N2).

MODES FOR CARRYING OUT THE INVENTION Example 1

A reassortant influenza virus is produced using the A/PR/8/34 influenzastrain as a backbone strain and the A/Brisbane/10/07-like orA/Perth/16/09-like influenza strain as vaccine strain. Inhibitory agents(e.g. siRNAs, PSOs or PMOs) are designed such that they preferentiallyreduce transcription and/or translation of the HA and/or NA gene(s) ofthe A/PR/8/34 strain.

The suitability of the inhibitory agents is tested by introducing theinhibitory agent into a culture host and subsequently co-infecting theculture host with the backbone strain and the vaccine strain. Protein isextracted from the infected cells and the preferential reduction of thebackbone strain's HA and/or NA protein levels is assessed by comparingthe protein levels of the vaccine strain's and the backbone strain's HAand/or NA proteins by quantitative Western blot analysis.

Reassortant influenza viruses are produced by introducing the inhibitoryagent into the culture host and infecting the culture host with thebackbone and the vaccine strain. The culture host is cultured underconditions suitable for producing the reassortant influenza virus.

Example 2

Inhibitory agents of the invention were selected by comparing theireffects on the growth of the backbone strain to their effects on thegrowth of the vaccine strain.

The following virus strains were tested:

-   -   A/PR/8/34 (the backbone strain); and    -   A/Victoria (H3N2) (the vaccine strain).

Although they were designed for inhibiting A/PR/8/34 in the presence ofA/Perth/16/09, the following siRNAs were tested:

-   -   HA2, HA7-HA12 and HA19 from Table 1 (targeting HA); and    -   NA4, NA6-NA9, NA11, NA12 and NA22 from Table 1 (targeting NA).

Experimental controls include no treatment (no TF no siRNA),transfection only (i.e. no siRNA; TF no siRNA) and control siRNAs (K1,K2) that do not target the virus.

The siRNAs were introduced into MDCK cells in parallel experiments. TheMDCK cells were subsequently infected with the virus, and the viraltiter was measured.

The results are shown in FIGS. 1 and 2. All tested siRNAs targeting HAshow reduction of A/PR/8/34 growth. siRNAs NA4, NA6, NA7, NA12 and NA22show reduction of A/PR/8/34 growth. siRNAs HA7, HA8, HA10, NA7, NA9,NA11 and NA12 do not significantly inhibit A/Victoria growth. Suitableinhibitory agents of the invention would block backbone strainreplication but allow propagation of the vaccine strain. Therefore,siRNAs HA7, HA8, HA10, NA7 and NA9 are suitable inhibitory agents of theinvention when using A/PR/8/34 and A/Victoria.

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

TABLE 1 siRNA sequences (antisense Strain strand) A/Puerto Rico/8/34 HAGGGCCUCUGUGUUAUUAUA SEQ ID NO: 49 UGGGCCUCUGUGUUAUUAU SEQ ID NO: 11UGAGACUCUUACCUUAUAC SEQ ID NO: 12 GGGCCUCUGUGUUAUUAUA SEQ ID NO: 13GACCCUCCUACUUGAUAAU SEQ ID NO: 14 GGGCCUUUAUCGUCUUUCU SEQ ID NO: 15CCGUUUACCUUUAGAUUAU SEQ ID NO: 16 CAGAGGGAAAGGUCUUAUA SEQ ID NO: 17CCCUCCUACUUGAUAAUGA SEQ ID NO: 18 CAGGAUGUAACAUCUUUGU SEQ ID NO: 19CUGGGUUUCAUUCUCUAGU SEQ ID NO: 20 NA CCUGAUUAAUCGGAUUAUA SEQ ID NO: 21CCCGAUAUAUGUCGUUUCU SEQ ID NO: 22 CGAAGACCCAACUUAAUUA SEQ ID NO: 23GCCUGAUUAAUCGGAUUAU SEQ ID NO: 24 CGGACACAUUUACCAAGUA SEQ ID NO: 25CGGGAAAUAAAGUACAAGA SEQ ID NO: 26 GCCGUUAAGUAGAGAAACA SEQ ID NO: 27CGCCUUCAAAGCAAGUUGU SEQ ID NO: 28 CAGACCAUCAGCCUGAUUA SEQ ID NO: 29CCCUGACAAUUCCUGUCUU SEQ ID NO: 30 CACCCGAUAUAUGUCGUUU SEQ ID NO: 31A/Ann Arbor/6/60 HA GUAAAGCUCUUUCAUUUCU SEQ ID NO: 32CGCCUCUUGUUUACGAUUA SEQ ID NO: 33 CCUCUUGUUUACGAUUAUU SEQ ID NO: 34CUCGUUAUUUAUGUUGUA SEQ ID NO: 35 CACGGUCUUACCAGGAUAU SEQ ID NO: 36GCCUCUUGUUUACGAUUAU SEQ ID NO: 37 GUCCGUUGAAGUUACUAAU SEQ ID NO: 38CGUGGUCUCAUACCUAAGU SEQ ID NO: 39 GGUCUUACCAGGAUAUAUU SEQ ID NO: 40 NACCGGGCAAUAUCUGUAUUU SEQ ID NO: 41 CGGGCAAUAUCUGUAUUUA SEQ ID NO: 42CCCACAAGGUAAAGUAAAU SEQ ID NO: 43 CCCGCUUGUAGUUAAAGUA SEQ ID NO: 44CGGCGUUACAGUUUAAUGU SEQ ID NO: 45 GGGAGUAGCUUGGGAUAAU SEQ ID NO: 46CGGCUAUGAUCUUAUGAUA SEQ ID NO: 47 CCUGGAGUUUGUCAUAACA SEQ ID NO: 48CGGUACACUUGGUUAUUAU SEQ ID NO: 50 A/Chile/1/83 HA CUGGUCUUAAAGUCUUUAUSEQ ID NO: 51 CCGUUUACCUUUAGAUUAU SEQ ID NO: 52 GGACCUGUAAACCUGUAUASEQ ID NO: 53 CCCUGUGUUAUUAUAAACU SEQ ID NO: 54 CUGGGUUUCAUUCUUUAGUSEQ ID NO: 55 GACCUGUAAACCUGUAUAU SEQ ID NO: 56 CUCCUUGACUCCCUUGUUASEQ ID NO: 57 CCCAUUUCUUAAGUUGUUU SEQ ID NO: 58 CCAGAAACGUCACGUCUUASEQ ID NO: 59 CAGGAUGUAACGUCUUUGU SEQ ID NO: 60 NA CUGGGUUCCACGAGAUAAUSEQ ID NO: 61 CCCGAUAUAUGUCGUUUCU SEQ ID NO: 62 CGAAGACCCAACUUAAUUASEQ ID NO: 63 GACCCAUUUAGUUUGUAUA SEQ ID NO: 64 GCCGUUAAGUAGAGAAACASEQ ID NO: 65 CGAGGUCUUUCCCUAAACU SEQ ID NO: 66 GGUUCCACGAGAUAAUUUASEQ ID NO: 67 CGCCUUCAAAGCAAGUUGU SEQ ID NO: 68 CCCAUUUAGUUUGUAUACASEQ ID NO: 69 CGGAGCAUGUCUUAGAAGU SEQ ID NO: 70siRNAs that differentially inhibit PR/8/34 relative to A/Perth/16/09* HATTTGGGATAATCATAAGTC SEQ ID NO: 71 HA1 TTTGTTGAATTCTTTACCC SEQ ID NO: 72HA2 TTCTGCACTGCAAAGATCC SEQ ID NO: 73 HA3 TTGATTCCAATTTCACTCCSEQ ID NO: 74 HA4 TTCTTTGGGAAATATTTCG SEQ ID NO: 75 HA5TAATCTCAGATGCATATTC SEQ ID NO: 76 HA6 TTCATTCTGATAGAGATTC SEQ ID NO: 77HA7 TTCACCTTGTTTGTAATCC SEQ ID NO: 78 HA8 TTTCTTACACTTTCCATGCSEQ ID NO: 79 HA9 TAGACCTCTGGATTGAATG SEQ ID NO: 80 HA10TACTTTCTCATACAGATTC SEQ ID NO: 81 HA11 TACACTCATGCATTGATGC SEQ ID NO: 82HA12 TTTGGTGTTTCTACAATGT SEQ ID NO: 83 HA13 TCAGCTTTGGGTATGAGCCSEQ ID NO: 84 HA14 TAGTCCTGTAACCATCCTC SEQ ID NO: 85 HA15ATTTCTTACACTTTCCATG SEQ ID NO: 86 HA16 TACTGTGTCAACAGTGTCG SEQ ID NO: 87HA17 TTACACTTTCCATGCATTC SEQ ID NO: 88 HA18 TTTGTAATCCCGTTAATGGSEQ ID NO: 89 HA19 ATAGAGATTCTGTTGTTCC SEQ ID NO: 90 HA20TTGGGATAATCATAAGTCC SEQ ID NO: 91 HA21 TTGAATTCTTTACCCACAG SEQ ID NO: 92HA22 TTTGTGTTGTGGTTGGGCC SEQ ID NO: 93 HA23 TTCTTCTCGAGTACTGTGTSEQ ID NO: 94 HA24 NA TACAGTATCACTATTCACG SEQ ID NO: 95 NA1TTTAATACAGCCACTGCTC SEQ ID NO: 96 NA2 ATTGATTTAGTAACCTTCC SEQ ID NO: 97NA3 TATCTGGACCTGAAATTCC SEQ ID NO: 98 NA4 TTGATTTAGTAACCTTCCCSEQ ID NO: 99 NA5 TTGAATTGAATGGCTAATC SEQ ID NO: 100 NA6TTGCTGTATATAGCCCACC SEQ ID NO: 101 NA7 TTGCCGGTTAATATCACTGSEQ ID NO: 102 NA8 TAACAGTCCCACTTGAATG SEQ ID NO: 103 NA9TTTGGTTGCATATTCCAGT SEQ ID NO: 104 NA10 TATTAGGCTAATTAGTCCGSEQ ID NO: 105 NA11 TTTGGAACCAATTCTTATG SEQ ID NO: 106 NA12ATCTACAGTATCACTATTC SEQ ID NO: 107 NA13 TACTTGTCAATGCTGAATGSEQ ID NO: 108 NA14 TTACTATCAGTCTCTGTCC SEQ ID NO: 109 NA15TTGACTTCCAGTTTGAATT SEQ ID NO: 110 NA16 ATTAATTCAACCCAGAAGCSEQ ID NO: 111 NA17 TCCTATTTGATAATCCAGG SEQ ID NO: 112 NA18TGAATTGAATGGCTAATCC SEQ ID NO: 113 NA19 TTCCAGTTTGAATTGAATGSEQ ID NO: 114 NA20 ATGGTTTCAGTTATTATGC SEQ ID NO: 115 NA21ATGTTGAACGAAACTTCCG SEQ ID NO: 116 NA22 ATTGCCACAACATCTTGCCSEQ ID NO: 117 NA23 TTGGAACCAATTCTTATGC SEQ ID NO: 118 NA24 *Thefollowing sequences are provided in DNA format and only the antisensestrand of each siRNA is shown. For the experiments, the inventors useddouble stranded RNAs (i.e. G, A, C and U ribonucleotides) based on thesequences below, and both siRNA strands contained 2 additional Unucleotidesas overhangs at the 3′ends.

TABLE 2 Strain Sequences HA A/PuertoGGGCCTCTGTGTTATTATAGGGGGGGTATAATAACACAGAGGCCC Rico/8/34 (SEQ ID NO: 119)TGGGCCTCTGTGTTATTATGGGGGGGATAATAACACAGAGGCCCA (SEQ ID NO: 120)A/Ann Arbor/6/60 GTAAAGCTCTTTCATTTCTGGGGGGGAGAAATGAAAGAGCTTTAC(SEQ ID NO: 121) CGCCTCTTGTTTACGATTAGGGGGGGTAATCGTAAACAAGAGGCG(SEQ ID NO: 122) A/Chile/1/83CTGGTCTTAAAGTCTTTATGGGGGGGATAAAGACTTTAAGACCAG (SEQ ID NO: 123)CCGTTTACCTTTAGATTATGGGGGGGATAATCTAAAGGTAAACGG (SEQ ID NO: 124) NAA/Puerto CCTGATTAATCGGATTATAGGGGGGGTATAATCCGATTAATCAGG Rico/8/34(SEQ ID NO: 125) CCCGATATATGTCGTTTCTGGGGGGGAGAAACGACATATATCGGG(SEQ ID NO: 126) A/Ann Arbor/6/60CCGGGCAATATCTGTATTTGGGGGGGAAATACAGATATTGCCCGG (SEQ ID NO: 127)CGGGCAATATCTGTATTTAGGGGGGGTAAATACAGATATTGCCCG (SEQ ID NO: 128)A/Chile/1/83 CTGGGTTCCACGAGATAATGGGGGGGATTATCTCGTGGAACCCAG(SEQ ID NO: 129) CCCGATATATGTCGTTTCTGGGGGGGAGAAACGACATATATCGGG(SEQ ID NO: 130)

DEPOSIT INFORMATION

A deposit of the microorganism MDCK 33016 (DSM ACC2219) was deposited onJun. 7, 1995 according to the Budapest Treaty in the InternationalDepository Authority DSM-Deutsche Sammlung Von Mikroorganismen andZellkulturen GmbH, Mascheroder Weg lb, D-38124 Braunschweig.

REFERENCES

-   [1] Rota et al. (1992) J Gen Virol 73:2737-42.-   [2] The Protein Protocols Handbook (ed. Walker). 2nd edition, 2002,    ISBN: 0-89603-940-2.-   [3] Miller et al. (2003) PNAS 100:7195-7200.-   [4] Ge et al. (2003) PNAS 100:2718-2723.-   [5] jura.wi.mit.edu/bioc/siRNAext-   [6] www.dharmacon.com/designcenter/designcenterpage.aspx-   [7] WO2009/030440.-   [8] Summerton and Weller (1997) Antisense Nucleic Acid Drug Dev.;    7(3):187-195.-   [9] Klee et al. (2005); Nucleic Acids Res. 1; 33.-   [10] WO2005/113756.-   [11] Kistner et al. (1998) Vaccine 16:960-8.-   [12] Kistner et al. (1999) Dev Biol Stand 98:101-110.-   [13] Bruhl et al. (2000) Vaccine 19:1149-58.-   [14] WO97/37000.-   [15] Brands et al. (1999) Dev Biol Stand 98:93-100.-   [16] Halperin et al. (2002) Vaccine 20:1240-7.-   [17] Tree et al. (2001) Vaccine 19:3444-50.-   [18] Kistner et al., (1998) Vaccine 16:960-8.-   [19] Kistner et al. (1999) Dev Biol Stand 98:101-110.-   [20] Bruhl et al. (2000) Vaccine 19:1149-58.-   [21] Pau et al. (2001) Vaccine 19:2716-21.-   [22] www.atcc.org-   [23] locus.umdnj.edu-   [24]WO03/076601.-   [25] WO2005/042728.-   [26] WO03/043415.-   [27] WO01/85938.-   [28] WO2006/108846.-   [29] WO97/37000.-   [30] Brands et al. (1999) Dev Biol Stand 98:93-100.-   [31] Halperin et al. (2002) Vaccine 20:1240-7.-   [32] EP-A-1260581 (WO01/64846).-   [33] WO2006/071563.-   [34] WO2005/113758.-   [35] WO03/023021.-   [36] WO03/023025.-   [37] WO97/37001.-   [38] WO2006/027698.-   [39] WO97/37001.-   [40] WO02/28422.-   [41] WO02/067983.-   [42] WO02/074336.-   [43] WO01/21151.-   [44] WO02/097072.-   [45] WO2005/113756.-   [46] Huckriede et al. (2003) Methods Enzymol 373:74-91.-   [47] Vaccines. (eds. Plotkin & Orenstein). 4th edition, 2004, ISBN:    0-7216-9688-0.-   [48] Treanor et al. (1996) J Infect Dis 173:1467-70.-   [49] Keitel et al. (1996) Clin Diagn Lab Immunol 3:507-10.-   [50] Herlocher et al. (2004) J Infect Dis 190(9):1627-30.-   [51] Le et al. (2005) Nature 437(7062):1108.-   [52] WO2008/068631.-   [53] Gennaro (2000) Remington: The Science and Practice of Pharmacy.    20th edition, ISBN: 0683306472.-   [54] Banzhoff (2000) Immunology Letters 71:91-96.-   [55] Nony et al. (2001) Vaccine 27:3645-51.-   [56] EP-B-0870508.-   [57] U.S. Pat. No. 5,948,410.-   [58] WO2007/052163.-   [59] WO2007/052061.-   [60] WO90/14837.-   [61] Podda & Del Giudice (2003) Expert Rev Vaccines 2:197-203.-   [62] Podda (2001) Vaccine 19: 2673-2680.-   [63] Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell    & Newman) Plenum Press 1995 (ISBN 0-306-44867-X).-   [64] Vaccine Adjuvants: Preparation Methods and Research Protocols    (Volume 42 of Methods in Molecular Medicine series). ISBN:    1-59259-083-7. Ed. O'Hagan.-   [65] WO2008/043774.-   [66] Allison & Byars (1992) Res Immunol 143:519-25.-   [67] Hariharan et al. (1995) Cancer Res 55:3486-9.-   [68] US-2007/014805.-   [69] US-2007/0191314.-   [70] Suli et al. (2004) Vaccine 22(25-26):3464-9.-   [71] WO95/11700.-   [72] U.S. Pat. No. 6,080,725.-   [73] WO2005/097181.-   [74] WO2006/113373.-   [75] Potter & Oxford (1979) Br Med Bull 35: 69-75.-   [76] Greenbaum et al. (2004) Vaccine 22:2566-77.-   [77] Zurbriggen et al. (2003) Expert Rev Vaccines 2:295-304.-   [78] Piascik (2003) J Am Pharm Assoc (Wash D.C.). 43:728-30.-   [79] Mann et al. (2004) Vaccine 22:2425-9.-   [80] Halperin et al. (1979) Am J Public Health 69:1247-50.-   [81] Herbert et al. (1979) J Infect Dis 140:234-8.-   [82] Chen et al. (2003) Vaccine 21:2830-6.-   [83] Current Protocols in Molecular Biology (F. M. Ausubel et al.,    eds., 1987) Supplement 30.-   [84] Smith & Waterman (1981) Adv. Appl. Math. 2: 482-489.

The invention claimed is:
 1. A method of preparing a reassortantinfluenza virus comprising the steps of (i) contacting a culture hostinfected with a vaccine strain and a backbone strain with a nucleic acidinhibitory agent wherein said inhibitory agent preferentially reducesthe transcription and/or translation of the hemagglutinin and/orneuraminidase genes of the backbone strain; and, (ii) culturing theculture host in order to produce the reassortant virus.
 2. The method ofclaim 1, further comprising the step (iii) of purifying the reassortantvirus obtained in step (ii).
 3. A method of preparing a reassortantinfluenza virus comprising the steps of (i) introducing into a culturehost that has been infected with a first influenza virus strain havingat least one target segment one or more expression construct(s) encodingthe target segment(s) from a second influenza virus strain; (ii)contacting the culture host with a nucleic acid inhibitory agent whichpreferentially reduces the transcription and/or translation of the firstinfluenza strain's target segment(s); (iii) culturing the culture hostin order to produce reassortant virus; and optionally (iv) purifying thevirus obtained in step (iii).
 4. The method of claim 1, furthercomprising the steps of (a) infecting a culture host with the virusobtained in step (ii) of claim 1; (b) culturing the host from step (a)to produce further virus; and (c) purifying the virus obtained in step(b).
 5. The method of claim 3, further comprising the steps of (a)infecting a culture host with a reassortant influenza virus obtained instep (iii) or (iv) of claim 3; (b) culturing the host from step (a) toproduce the virus; and optionally (c) purifying the virus obtained instep (b).
 6. A method of preparing a vaccine, comprising steps of (a)preparing a reassortant virus by the method of claim 1 and (b) preparinga vaccine from the reassortant virus.
 7. The method of claim 1, whereinthe culture host is an embryonated hen egg.
 8. The method of claim 1,wherein the culture host is a mammalian cell.
 9. The method of claim 8,wherein the cell is an MDCK, Vero or PerC6 cell.
 10. The method of claim8, wherein the cell grows adherently.
 11. The method of claim 8, whereinthe cell grows in suspension.
 12. The method of claim 11, wherein thecell is of the cell line MDCK 33016 (DSM ACC2219).
 13. The method ofclaim 4, wherein step (c) involves inactivating the virus.
 14. Themethod of claim 6, wherein the vaccine is a whole virion vaccine. 15.The method of claim 6, wherein the vaccine is a split virion vaccine.16. The method of claim 6, wherein the vaccine is a surface antigenvaccine.
 17. The method of claim 6, wherein the vaccine is a virosomalvaccine.
 18. The method of claim 6, wherein the vaccine contains lessthan 10 ng of residual host cell DNA per dose.
 19. The method of claim 1wherein at least one of the influenza strains is of the H1, H2, H5, H7or H9 subtype.
 20. The method of claim 19, wherein at least one of thestrains is a H1N1, H5N1, H5N3, H9N2, H2N2, H7N1 or H7N7 strain.
 21. Themethod of claim 1, wherein one of the influenza strains is a high-growthstrain.
 22. The method of claim 19, wherein at least one influenzastrain is selected from the group consisting of A/Puerto Rico/8/34,A/Ann Arbor/6/60, B/Ann Arbor/1/66, A/Chile/1/83 and A/California/04/09.23. A cell for preparing a reassortant influenza virus comprisingexpression construct(s) encoding: (i) all eight viral segments of afirst influenza A or B virus genome; (ii) at least one target segment ofa second influenza A or B virus genome, wherein the second influenzastrain's target segment(s) differs in sequence from the target segmentof the first influenza strain; and (iii) a nucleic acid inhibitory agentwherein said inhibitory agent preferentially reduces transcriptionand/or translation of the target segment(s) of the first influenzastrain.
 24. The method of claim 1, wherein the nucleic acid inhibitoryagent is an antisense oligonucleotide.
 25. The method of claim 1,wherein the nucleic acid inhibitory agent is a synthetic antisenseoligomer.
 26. The method of claim 1, wherein the nucleic acid inhibitoryagent is a phosphorothioate oligomer (PMO).
 27. The method of claim 1,wherein the nucleic acid inhibitory agent is a phosphorodiamidatemorpholino oligomer (PSO).
 28. The method of claim 1, wherein thenucleic acid inhibitory agent is selected from the group consisting ofshort interfering RNAs (siRNA), double-stranded RNAs (dsRNA), micro-RNAs(miRNA), short hairpin RNAs (shRNA), and small interfering DNAs (siDNA).